Removal of Estrogenic Compounds from Filtered Secondary

A 2 L enzymatic membrane reactor (EMR) was successfully operated for 100 h with ... low effort has been devoted for its implementation in a continuous...
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Removal of Estrogenic Compounds from Filtered Secondary Wastewater Effluent in a Continuous Enzymatic Membrane Reactor. Identification of Biotransformation Products Lucia Lloret,* Gemma Eibes, M. Teresa Moreira, Gumersindo Feijoo, and Juan M. Lema Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain S Supporting Information *

ABSTRACT: In the present study, a novel and efficient technology based on the use of an oxidative enzyme was developed to perform the continuous removal of estrogenic compounds from polluted wastewaters. A 2 L enzymatic membrane reactor (EMR) was successfully operated for 100 h with minimal requirements of laccase for the transformation of estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)from both buffer solution and real wastewater (filtered secondary effluent). When the experiments were performed at high and low concentrations of the target compounds, 4 mg/L and 100 μg/L, not only high removal yields (80−100%) but also outstanding reduction of estrogenicity (about 84−95%) were attained. When the EMR was applied for the treatment of municipal wastewaters with real environmental concentrations of the different compounds (0.29−1.52 ng/ L), excellent results were also achieved indicating the high efficiency and potential of the enzymatic reactor system. A second goal of this study relied on the identification of the transformation products to elucidate the catalytic mechanism of estrogens’ transformation by laccase. The formation of dimers and trimers of E1, E2, and EE2, as well as the decomposition of E2 into E1 by laccase-catalyzed treatment, has been demonstrated by liquid chromatography atmospheric pressure chemical ionization (LC-APCI) analysis and confirmed by determination of accurate masses through liquid chromatography electrospray time-of-flight mass spectrometry (LC-ESI-TOF). Dimeric products of E2 and EE2 were found even when operating at environmental concentrations. Moreover, the reaction pathways of laccase-catalyzed transformation of E2 were proposed.

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INTRODUCTION Over the past decades, public concern about the environmental impact of steroid estrogens has grown due to their negative effects associated with the disruption of the endocrine system in humans and animals.1,2 The presence of this type of compounds in the environment has been mainly attributed to their incomplete removal by conventional biological and physicochemical processes in wastewaters treatment plants (WWTP),1 being the natural compounds estrone (E1) and 17β-estradiol (E2) and the synthetic compound 17αethinylestradiol (EE2) the major contributors to the estrogenic activity detected in the WWTP effluents.3 Therefore, recent works have been focused on the development of advanced oxidation processes such as ozonation and chlorination. These alternatives provide satisfactory removal yields although they present important disadvantages such as high costs and formation of byproducts with unknown estrogenicity, which may be even higher than that of the original compound.4−6 Hence, an effective method is required to fulfill the challenge of their removal. In this way, the enzymatic treatment is a potential alternative for the removal of estrogens due to its low energy requirements and easy control and operation.7 Indeed, several previous © 2013 American Chemical Society

researchers have studied the transformation of E1, E2, and EE2 by ligninolytic enzymes, being laccase (EC 1.10.3.2, benzenodiol/oxygen oxidoreductase) the most widely applied biocatalyst due to its potential ability to degrade recalcitrant compounds and its simple catalytic mechanism using oxygen as final electron acceptor.8 Previous works reported significant removal yields of both the target compounds and their estrogenic activities.1,4,7,9−14 Nevertheless, most of those experiments were conducted at relatively high concentrations in synthetic media, whereas only a few investigations assessed the removal of estrogens in real wastewaters.1 Furthermore, research has been focused in the operation in batch mode, whereas relative low effort has been devoted for its implementation in a continuous process. Thus, the design and operation of a novel technology for the continuous application of enzymatic remediation under more realistic conditions is still a pending objective. Received: Revised: Accepted: Published: 4536

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Table 1. Sequence of Experiments Performed by Operating Continuously the EMR with a HRT of 4 h and an Initial Laccase Activity of 100 U/L experiment experiment experiment experiment

1 2 3 4

matrix

estrogens concentration

feed addition rate

phosphate buffer solution (0.1 M, pH 7) phosphate buffer solution (0.1 M, pH 7) spiked real wastewater real wastewater

4 mg/L 100 μg/L 100 μg/L environmental concentrations: 0.29−1.52 ng/L

1 mg/(L·h) 25 μg/(L·h) 25 μg/(L·h) 0.07−0.38 ng/(L·h)

was based on a previous design and consisted of a tank reactor coupled to an ultrafiltration polyethersulfone membrane (Prep/ Scale-TFF Millipore) with a nominal molecular weight cutoff of 10 kDa.6 The influent containing the mixture of the estrogens was continuously fed into the tank by a peristaltic pump, whereas the laccase was added in a single initial pulse. An additional pump was used to circulate the reaction solution from the tank to the membrane where the enzyme was retained and continuously recycled to the reactor at a recycling/feed flow ratio 12:1. A valve located in the membrane module permitted the control of both effluent and recycling flow rates. PTFE tubing was used to avoid the adsorption of the target compounds to the inner surface of the tubing. Once the feasibility of a lab-scale reactor of 250 mL of volumen has been previously demonstrated,6 a larger-scale bioreactor (2 L) was used in the current work (Biostat MD, B. Braun-Biotech International), which was equipped with pH, temperature, and pO2 sensors as well as with mechanical stirring. An electrovalve located at the end of a flexible membrane tube controlled by a cyclic timer was used to inject oxygen with a pulsing flow of 1 bar for 30 s each 30 min of operation time. Temperature was maintained at 26 °C by circulating thermostatted water through the reactor chamber and the reaction mixture was continuously stirred at 250 rpm. A scheme of the bioreactor is shown in Figure S1 of the Supporting Information. The hydraulic residence time (HRT) was 4 h, which meant an inlet flow rate of 8.3 mL/min. Besides, an initial laccase activity of 100 U/L was assayed. The reactor was operated for 100 h to demonstrate the viability of the technology and the stability of both the membrane and the biocatalyst. Samples were withdrawn at different periods from the reaction vessel to measure laccase activity as well as from the effluent to determine both the estrogens concentration and the estrogenic activity. The performed experiments and the corresponding operational conditions are presented in Table 1. The performance of the technology was evaluated in terms of the removal percentage (%) of both target compounds and estrogenicity as well as by the estrogens removal rates (mg, μg, or ng degraded per volume of reactor and time). Corresponding controls lacking laccase were performed under the conditions examined in each removal experiment. The real wastewater used was collected from the outlet of the secondary clarifier of the municipal wastewater treatment plant of Calo-Milladoiro (Ames, Spain). This water was filtered (0.45 μm) to remove particulate matter and suspended solids, which could contain bacteria and other microorganisms, to avoid undefined biological transformation or even adsorption of the target compounds, and thus examining the sole role of the laccase, and then it was stored at 4 °C until its use. The wastewater was analyzed according to Standard Methods20 and the main characteristics are summarized in Table S1 of the Supporting Information.

We have previously developed a lab-scale enzymatic membrane reactor (EMR) based on a continuous stirred tank reactor coupled to an ultrafiltration membrane for the continuous elimination of E1 and E2 at high initial concentrations from a buffer solution by high doses of laccase obtaining promising results.6 Besides, this technology presents several advantages such as reuse of the biocatalyst, easy addition of fresh enzyme in case of deactivation, high flow rates, reduced energy requirements, simple operation and control, and straightforward scale-up.15,16 Thus, this enzymatic reactor can be particularly useful when performing the continuous treatment of real wastewaters by laccase at a larger scale (2 L EMR) and much lower initial concentrations of estrogens and enzyme. Indeed, integrate systems including filtration are considered nowadays one of the most promising technologies used for the advanced treatment of secondary effluents, being both micro- and ultrafiltration membranes techniques widely used as tertiary treatments.17−19 Furthermore, an evident lack of knowledge exists with regard to the identification of laccase-catalyzed transformation products of estrogens, in spite of this enzyme being so extensively tested over the past years. The elimination of estrogens has been assumed to occur by means of polymerization reactions.1,13,14 However, only few works supported this hypothesis by means of the corresponding experimental assays and analytical techniques: Nicotra et al. and Tanaka et al. reported the formation of dimers of E2 and EE2, respectively;11,12 whereas no other degradation products were identified and byproducts resulting from laccase-catalyzed oxidation of E1 have not been characterized up to date. To sum up, the key objective of this research was to establish and operate an efficient laccase-based bioreactor for the continuous removal of estrogenic compounds present in real wastewaters. The EMR was applied for the treatment of both buffer solution and real wastewater (filtered secondary effluent) at high and low as well as at environmental conditions. A second goal relied on the identification of transformation products and the proposal of reaction pathways to elucidate the catalytic mechanism of estrogens’ transformation by laccase.

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EXPERIMENTAL SECTION Chemicals. All chemicals were of analytical grade. Estrogens: E1, E2 and EE2, were obtained from Sigma-Aldrich (USA). Deuterated 17β-estradiol (E2-d4), deuterium was introduced in positions 2, 4, and 16, was purchased from Cambridge Isotope Laboratories (USA). 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was supplied from Fluka (USA). Stock solutions of E1, E2, EE2, and E2-d4 were prepared in methanol (J.T. Baker, HPLC grade, 99.8%). Commercial laccase from Myceliophthora thermophila (56 kDa) was supplied by Novozymes (Denmark). Reactor Design and Experimental Procedure. A continuous EMR was operated for the enzymatic removal of natural (E1 and E2) and synthetic (EE2) estrogens. The system 4537

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Enzymatic Activity. Laccase activity was determined by measuring the oxidation of 5 mM ABTS to its cation radical (ABTS+) at 436 nm in 100 mM sodium acetate buffer, pH 5, and 30 °C (ε436 = 29 300 M−1·cm−1) using a Shimadzu UV1603 spectrophotometer. One unit (U) of activity was defined as the amount of enzyme forming 1 μmol of ABTS+ per min. Evaluation of Estrogenic Activity. The estrogenic activities of the inlet and outlet streams of the reactor were measured by LYES (lyticase yeast estrogen screen) assay using recombinant yeast Saccharomyces cerevisae. This method has been adapted from one developed by Schultis and Metzger and described elsewhere.6,21 Estrogens Analysis. All of the analytical methods applied are detailed in the Supporting Information. The determination of the estrogens concentrations in experiment 1 (high initial estrogens concentration in buffer solution) was carried out by high performance liquid chromatography (HPLC) using a diode array detector. The samples withdrawn during experiments 2 and 3 (low initial concentrations in buffer solution or real wastewater) were analyzed by gas chromatography−mass spectrometry (GC−MS). First, samples of 20 mL were acidified at pH 2, diluted in distilled water (pH 2 adjusted with HCl) until a final volume of 100 mL, and filtered (0.45 μm). Then, the solid phase extraction (SPE) was carried out with 60 mg OASIS HLB cartridges (Water closet, USA) previously conditioned with 3 mL of ethyl acetate, 3 mL of methanol, and 3 mL of distilled water (pH 2). The cartridges were then dried with nitrogen for 45 min and eluted with 3 mL of ethyl acetate. BSTFA (N,O-bis(trimethylsisyl)trifluoroacetamide) was added for the derivatization of the species. Afterward, the resulting samples were analyzed by GC−MS. The concentrations of estrogens present in the real wastewater samples used as well as in the samples withdrawn during experiment 4 (environmental concentrations) were determined by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MSMS). Aiming to deeply concentrate the samples, volumes of 2 L (wastewater samples) or 5 L (samples after laccase-catalyzed treatment) were collected for their subsequent extraction and concentration to 3 mL by SPE as described above. Then, 2 mL of the resulting samples were evaporated under gentle stream of nitrogen prior to resuspension in methanol; finally, further concentration by nitrogen was conducted until obtaining 50 μL samples. Concentrations of estrogens quantified in these samples were corroborated using E2-d4 as an internal standard aiming to correct for matrix effects as indicated in the Supporting Information. Identification of Laccase-Catalyzed Reaction Products. With the objective of identifying the oxidation products and establishing the transformation pathways, experiments were conducted in batch reactors of 250 mL. Each reactor contained 100 mL of 5 mg/L of each estrogen in distilled water. An initial laccase activity of 2000 U/L was used aiming to ensure the degradation of the compounds and the consequent formation of reaction products at significant concentrations. Each estrogen was assessed separately to identify the corresponding transformation products. The reaction was conducted for 24 h and then the mixture was acidified to a final pH of 2 to inactivate the enzyme. These acidified samples as well as acidified samples corresponding to time 0 (before the enzyme addition and analogous to samples after 24 h of controls lacking enzyme)

and a blank with enzyme (after 24 of incubation in buffer solution) were analyzed by the LC-APCI method used for the quantification of estrogens. However, for this study the mass spectrometer was used in the full scan mode rather than in the tandem MS-MS mode, and the range selected for the mass spectrometer full scan was between m/z ratios of 50 and 900. Furthermore, samples were analyzed by liquid chromatography electrospray time-of-flight mass spectrometry (LC-ESITOF) in the negative mode by the method detailed in the Supporting Information aiming to determine accurate masses of the biotransformation products to confirm their proposed structures.

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RESULTS AND DISCUSSION Continuous Removal of Estrogens in an Enzymatic Membrane Reactor. A 2 L EMR was proposed for the continuous transformation of estrogens by free laccase once the feasibility of a similar 250 mL bioreactor for the removal of E1 and E2 by 500 U/L of laccase had been previously reported.6 In the present work, the removal of not only these natural compounds but also a synthetic estrogen (EE2) was attempted, and initial laccase activity was decreased to 100 U/L to reduce the enzyme requirements. The experimental conditions of the different performed assays are detailed in Table 1. First, the EMR was fed with a buffer solution (phosphate buffer solution, pH 7) containing high estrogens concentration (4 mg/L each) (experiment 1) to study the effect of the change of scale and the reduction of laccase activity. Removal percentages of 80, 87, and 85% of E1, E2, and EE2 respectively were attained under steady-state conditions, which implied degradation rates in the range of 0.80−0.87 mg/(L·h) as shown in Figure S2 of the Supporting Information. The use of a much lower laccase activity slightly decreased the removal yields by 15%: degradation percentages between 96 and 98% were previously found using 500 U/L.6 Moreover, no biocatalyst inactivation was detected over the 100 h of operation and the estrogenicity was reduced by 84%. Results of the corresponding control assay verified that removal of the target compounds was caused only by laccase action: the average of the estrogens outlet rate (determined as the concentration in the effluent per residence time) matched with the feed addition rate as also occurred in the following experiments. Experiment 2 was conducted with 100 μg/L (each) of the estrogens in buffer solution as an attempt to work at lower concentrations and closer to environmental levels. The results evidenced the capability of the enzymatic technology to remove these pollutants even at such low concentration obtaining higher efficiencies than those in the previous experiment: E1 was eliminated by 88% (removal rate 22 μg/(L·h)) and E2 and EE2 were not detected (below detection limits), which means removal percentages of up to 99 and 94%, respectively (Figure S3 of the Supporting Information). Moreover, a reduction of 95% of estrogenic activity was found and the biocatalyst retained its total initial activity after 100 h. With the goal of studying the matrix effect to investigate the possibility of the real implementation of this technology, experiment 3 was performed with real wastewater (filtered secondary effluent) previously spiked with 100 μg/L of the estrogens. The results of the variables monitored during the operation are shown in Figure 1. As it can be observed, despite a partial inactivation of laccase (20%) during the first hours, probably due to the constituents of the wastewater, an activity close to 80 U/L was constantly maintained after 100 h of 4538

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real wastewaters at environmental concentrations and under oxygenated conditions. Some previous works have been also focused on the enzymatic removal of estrogens from real wastewaters.1,10 However, those investigations used spiked wastewaters, whereas the capability of the laccase system to remove environmental concentration of estrogens was still an important challenge. Furthermore, those previous works only tested the efficiency of the enzymatic system in batch operation, whereas scarce references dealing with the enzymatic continuous process are available. Moreover, a relatively low initial enzyme activity was used in the current research (100 U/L) in comparison with those previously needed to attain similar results. For instance, Auriol et al. reported the total transformation of the pollutants using 20 000 U/L of laccase or alternatively 5000 U/L in the presence of 1-hydroxybenzotriazole (HBT) as mediator.1 Overall, the results reported here are encouraging as they present an innovative technology to remove natural and synthetic estrogens found in sewage effluents and thus demonstrated the potential implementation of this novel technology as an alternative advanced oxidation process in conventional treatment plants. As far as we know, this is the first time that an enzymatic bioreactor was successfully developed and operated in continuous mode for the treatment of real municipal wastewaters at both high and low concentrations as well as under environmentally realistic conditions. Identification of Biotransformation Products. Although we have recently demonstrated the formation of metabolites by the laccase-catalyzed oxidation of E1 and E2 using GC−MS, the identification of these reaction products was not possible.6 However, some authors suggested that the removal of estrogens could occur by polymerization because these compounds possess a parasubstituted phenol structure and the enzyme may catalyze the oxidative coupling of phenolic compounds.7,11,12 This assumption has been stated in several investigations dealing with laccase-catalyzed treatment of estrogens to explain the disappearance of the target compounds and the difficulties in characterizing the reaction products. Nonetheless, only few works demonstrated this hypothesis by experimental assays and the appropriate analytical methods. For instance, the formation of dimers of E2 by laccase action has been previously reported by Nicotra et al. and Tanaka et al. reported the formation of a single dimer of EE2 by laccase-mediated treatment.11,12 With the goal of verifying the estrogens’ radical coupling and characterizing the laccase-catalyzed reaction products of E1, E2, and EE2, a further study was conducted in the present work using an LC-APCI system coupled to a tandem mass spectrometer operated in the full scan mode, which allowed the detection of products with high m/z ratio. A concentration of 5 mg/L was selected for the batch assays to favor the extent of the reaction to establish the complete laccase-catalyzed transformation pathways. All precursor ions in APCI positive were the results of a simple protonation. Moreover, in the cases of E2 and EE2 the analytes underwent a loss of water in the source as reported by Vanderford et al. when optimizing the analytical method to determine estrogens.28 Thus, the compounds based on E2 and EE2 were seen as [M + H −H2O] in the first quadrupole of the mass spectrometer (Q1), and [M + H] in the case of E1. The use of the Q1 under full scan monitoring in the range m/z 50− 900 revealed new peaks in total ion chromatograms (TIC) of the samples treated with laccase for 24 h. Although the

Figure 1. Time course of E1, E2, and EE2 degradation rates, laccase activity, and reduction of estrogenicity during the operation of the EMR for the treatment of real wastewater containing 100 μg/L of each estrogen (feed addition rate 25 μg/(L·h)) by 100 U/L of initial laccase activity (experiment 3), and average of estrogens outlet rate during the corresponding control assay lacking laccase.

operation until the bioreactor was stopped. Furthermore, high removal percentages of the estrogens were achieved: 80−92%, which implies degradation rates of 20−23 μg/(L·h), under steady-state conditions. This loss of removal efficiencies observed when comparing both assays with buffer solution and real wastewater may be caused by the slight biocatalyst inactivation and/or by the presence of other compounds, which could compete with the target compounds for the enzyme. As expected, the lower oxidation yields led to diminished estrogenicity reduction, although still a significant decrease was detected (90%). Consequently, the potential detrimental constituents of the real matrix (colloidal particles, organic matter, nitrogen-based compounds, etc.) exerted low impact on the removal yields and on the enzyme activity. Promising performance, stability, and catalytic efficiency of this technology were demonstrated evidencing its noticeable applicability on the treatment of real wastewaters. Indeed, although composite fouling (i.e., a combination of biofouling and inorganic fouling) often occurs in membrane systems because of the nature of wastewaters,22 neither fouling nor changes in the membrane pressure (1−1.5 bar) were detected here. In experiment 4, the capability of the system to remove the estrogens from wastewaters at real environmental concentrations was assessed. In this case, the bioreactor was fed with real nonspiked municipal wastewater containing the estrogens at real environmental concentrations of 0.29−1.52 ng/L (Table 1). Under these conditions, E1 was removed by 98% (degradation rate 0.37 ng/(L·h)), whereas E2 and EE2 were not detected in the effluent, which corresponds to oxidation percentages up to 97 and 99%, respectively (removal rates 0.07 and 0.18 ng/(L·h)). Thus, excellent results were found in spite of the possible presence of compounds, which also may act as laccase substrates such as phenols. Few authors reported the oxidation of E2 under abiotic conditions by various mechanisms such as by manganese oxides or through nitration in the presence of high nitrate concentrations.23,24 Also, Marfil-Vega et al. demonstrated the abiotic transformation of E1, E2, and EE2 in the presence of model vegetable matter and the important influence of the molecular oxygen during that mechanism.25−27 Anyhow, control assays corroborated the only implication of the laccase on the estrogenic compounds removal even when working with 4539

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Moreover, three new peaks were observed at retention times of 9.7, 10.9, and 12.2 min. These products were identified as dimers of E2 due to their molecular ions: dimer of E2 would have a MW of 542 (mass of E2 272 × 2 − 2H = 542); however, the compounds are seen as [M + H − H2O] in the Q1, therefore the molecular ion would be 525, as appeared in the spectrum of the E2 dimer I (part B of Figure S5 of the Supporting Information). Also, two trimers of E2 (MW 812) appeared at retention times 13.0 and 13.6 min and presented a molecular ion at a m/z of 795 (part C of Figure S5 of the Supporting Information). These results are in agreement with those reported by Mao et al., who demonstrated the formation of dimers and trimers, as well as E1, after the enzyme-mediated transformation of E2 using lignin peroxidase.33−35 The formation of dimers and trimers was also demonstrated by analyzing the new peaks in the TIC corresponding to EE2 (part A of Figure S6 of the Supporting Information). Two dimers were observed at retention times of 8.9 and 9.6 min, which have MW of 590 (mass of EE2 296 × 2 −2H = 590) although they presented a molecular ion of 573 (590 + H − H2O) as observed in the spectrum of the EE2 dimer I (part B of Figure S6 of the Supporting Information). Besides, two trimers of EE2 (MW 884) were found at 10.9 and 11.8 min of retention time and presented a molecular ion at m/z of 867 (part C of Figure S6 of the Supporting Information). A new peak was also detected at a retention time of 7.7 min with a molecular ion of 295 (Table 2). Previous authors reported hydroxylation reactions from EE2 by fungi and algae and in some cases a subsequent methoxylation of the hydroxyl derivate.36 In this way, this compound might also correspond to a hydroxylated product of EE2: its MW would be 312 (mass of EE2 296 − H + OH = 312), and it would be seen as 295 (312 + H − H2O). However, a further study should be conducted to ensure this premise. Afterward, samples were analyzed by LC-ESI-TOF to obtain accurate masses information of the biotransformation products. Considering the chemical formula of E1, E2, and EE2: C18H22O2, C18H24O2, and C20H24O2 respectively the dimers and trimers of these target compounds would be: C36H42O4 and C54H62O6, C36H46O4 and C54H68O6, and C40H46O4 and C60H68O6, respectively. Once ESI was used as ionization source in negative mode, parent compounds were deprotonated and seen as [M-H]. As observed in Table S5 of the Supporting Information, chemical formulas of the detected biotransformation products matched with deprotonated dimers and trimers of the estrogens, and also E1 was detected as an E2 product. Furthermore, experimental accurate masses were in agreement with the calculated ones with errors varying from −5.2 to 4.1 ppm: two E1 dimers were found to have accurate masses of 537.3037 and 537.3038, and E2 and EE2 dimers and trimers had masses in the ranges 541.3315−541.3334 and 811.4942− 811.4954 and 589.3312−589.3330 and 883.4949−883.4963, respectively (deprotonated compounds). Also, these values fitted those found in the literature.37,38 A larger number of dimeric and trimeric compounds were detected by LC-ESITOF in comparison with LC-APCI analysis probably because of a higher sensitivity of the method. The novelty of this investigation relies on the successful characterization of different dimers not only of E2 and EE2 but also of E1. Additionally, the formation of E2 and EE2 trimers as well as the transformation of E2 into E1 by laccase-catalyzed treatment was demonstrated for the first time.

identification of some of the products was not possible, most of them were characterized by their molecular weights (MW) as follows and as indicated in Table 2. Table 2. Characterization of the Products Detected by LCAPCI Formed after 24 h of E1, E2, or EE2 LaccaseCatalyzed Transformation, Observed in the Corresponding TIC after Subtracting the Signals of the Blank and Time 0 Samples parent compound

retention time (min)

molecular ion

molecular weight

suggested product

E1

8.1 13.7 7.9 9.7 10.9 12.2 13.0 13.6 7.7 8.9 9.6 10.9 11.8

269 539 271 525 525 525 795 795 295 573 573 867 867

268 538 270 542 542 542 812 812 312 590 590 884 884

not identified E1 dimer E1 E2 dimer I E2 dimer II E2 dimer III E2 trimer I E2 trimer II not identified EE2 dimer I EE2 dimer II EE2 trimer I EE2 trimer II

E2

EE2

In the case of E1, some new peaks were detected in the TIC corresponding to 24 h of laccase-catalyzed transformation (part A of Figure S4 of the Supporting Information). The first peaks displayed in the TIC (not indicated by their corresponding retention times) were also observed in the blank or time 0 samples and thus were not considered as reaction products. Therefore, only the peaks marked in the figures are supposed to be reaction products and were identified as shown in Table 2. A new product was observed at retention time 13.7 min and showed a m/z of 539. This compound could be an E1 dimer with MW 538: mass of E1 270 × 2 −2H = 538, and after protonation the molecular ion would be 539, as shown in the corresponding spectrum (part B of Figure S4 of the Supporting Information). Another compound with a molecular ion at m/z 269 was detected at a retention time of 8.1 min, although its identification was not possible. Regarding E2, different new peaks were observed in the TIC after subtracting the signal of the blank and time 0 samples (part A of Figure S5 of the Supporting Information) and were identified as indicated in Table 2. First, the results revealed a new compound with m/z 271 at a retention time of 7.9 min. This compound was presumed to be E1 formed upon oxidation of E2 because its spectrum corresponded to that of E1 standard. It could explain the apparent lower removal of E1 in comparison to E2 and EE2 when a mixture of the three compounds was treated by laccase in the EMR. Other authors also reported the transformation of E2 to E1 by other different treatments such as activated sludge from sewage treatment plants or nitrifying activated sludge.29 Nevertheless, this is the first time that E1 is characterized as an E2 transformation product by laccase. Furthermore, it is known that transformation of E2 into E1 is a quite unspecific oxidation, which can be carried out by many bacteria.30−32 However, although these assays were not run under sterile conditions, the corresponding controls lacking laccase discarded bacterial transformation of the target compounds. 4540

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Figure 2. Reaction products structures and proposed reaction pathways of laccase-catalyzed transformation of E2.

The formation of dimers would be also expected after the treatment of environmental concentrations considering the findings of previous investigations.33,38 Although the identification by LC-APCI of coupling products in the samples

collected from the EMR effluent fed with secondary effluent aiming to verify that assumption was not possible, probably due to the detection limits of the instrument, dimers of E2 and EE2 were successfully identified by LC-ESI-TOF: products with 4541

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accurate masses of 541.3343 and 541.3342 (C36H45O4) and 589.3330 (C40H45O4) respectively were found with relative errors from −3.7 to −1.2 ppm. Although the possibility of other reaction mechanisms could not be discarded, these results indicated that laccase-catalyzed radical coupling reactions occur even at such low concentrations. Proposed Reaction Pathways. As mentioned, the formation of dimers and trimers suggests the elimination via radical coupling reactions by laccase-catalyzed oxidation of the substrates to generate free radicals, which may couple covalently to each other subsequently. In fact, the products followed the pattern of nMW − 2(n − 1), where n is the number of monomers and MW the mass of the parent compound. However, it is interesting to highlight that, in the case of E1 and E2, other species having smaller MW than the initial compounds were found, which may indicate that radical coupling was not the only transformation reaction, but also different degradation mechanisms are involved. Reaction pathways were proposed for E2 because all of the products detected by LC-APCI after 24 h of laccase-catalyzed treatment of that compound were identified. The suggested products’ structures and reactions pathways are schematized in Figure 2. Regarding the oxidative radical−radical coupling, the reaction is initiated by the laccase-catalyzed formation of the primary oxidation product by abstracting one electron from the −OH group of the original molecule. Thus, the free radical is formed and the unpaired electron may delocalize through resonance to the respective conjugated positions (E2 radical intermediates, compounds 1−3). Thereafter, the subsequent covalent bonding between radical intermediates could occur through C−C or C− O bond formation. Mao et al. reported that oxygen atoms have higher charges and lower spin density than carbon atoms making bond formation at these sites kinetically less favorable.35 Anyhow, both possibilities were considered and the possible structures of the dimers are indicated as C−O and C−C dimeric products in Figure 2 (compounds 4−7). Because of the remaining laccase activity and that the coupling products are still substrates of the enzyme due to their phenolic groups, a radical coupling reaction can be further performed. Thus, the abstraction of another electron from one of the −OH groups of the dimeric products would occur. Once different dimeric products may have been formed and they present various −OH groups, there exist several possibilities of forming dimer radical intermediates. For instance, it is indicated in Figure 2 the radical intermediates formed from the C−O (compound 4) and C−C dimer (compound 6) products resulting from the abstraction of one electron of the −OH groups indicated (marked with asterisks) to form the corresponding oxygen radicals that can delocalize to carbonlocated radicals (intermediates 8 and 9 for the C−O dimer and intermediates 10 and 11 for C−C dimer). Afterward, the presence of radical intermediates of E2 in the reaction medium could lead to the formation of E2 trimers via radical−radical coupling mechanism. As an example of possible E2 trimers formed, the products resulted from the covalent bonding between the second radical form of each pair of dimer radical intermediates (compounds 9 and 11) and both radical forms 1 and 2 of E2 are shown (compounds 12−15). To our knowledge, the reaction pathways of laccase-catalyzed transformation of an estrogen including dimers, trimers, and E1 as a product of E2 degradation have been proposed and demonstrated in this study for the first time.

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ASSOCIATED CONTENT

S Supporting Information *

(1) Scheme of the EMR used, (2) characteristics of the filtered secondary effluent used, (3) HPLC method for the analysis of estrogens at high concentration, (4) GC−MS method for the analysis of estrogens at low concentrations, (5) LC-APCI-MSMS method for the analysis of estrogens at environmental concentrations, (6) results corresponding to experiments 1−2, (7) TIC of 24 h samples and mass spectra of transformation products detected by LC-APCI, (8) determination of accurate masses by LC-ESI-TOF. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +34881816771 +; fax: +34881816702; e-mail: lucia. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Spanish Ministry of Science and Innovation (MICINN, CTQ2010-20258). The authors belong to the Galician Competitive Research Group GRC2010/37. L. Lloret thanks the Spanish Ministry of Education for the FPU grant (AP2008-01954). G. Eibes thanks the Xunta de Galicia for an Angeles Alvariño contract.

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REFERENCES

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